Project Summary Zebrafish (Danio rerio) are an attractive vertebrate model for human disease and neuronal function because they possess brains simple enough to carry out fundamental studies of neuronal function, yet complex enough to provide pathways that are, in many ways, analogous to those in the human brain. The analysis of how neurons release neurotransmitters, such as dopamine, is essential for the study of human neurological disease and basic neuronal function in zebrafish. Published and preliminary results demonstrate the feasibility of measuring evoked and naturally occurring dopamine release in adult zebrafish with fast-scan cyclic voltammetry (FSCV) at carbon- fiber microelectrodes. However, the lack of a method to image neurons of interest while electrochemically measuring release presents a roadblock because the opaque environment of the living adult zebrafish brain makes the accurate and reliable analysis of remotely-evoked and spontaneously-occurring neurotransmitter release nearly impossible. Therefore, a critical need exists to combine FSCV with powerful imaging methods to help guide electrode positioning and as well as to optically measure neuronal firing in the intact, whole zebrafish adult brain. To solve this problem, this project will combine sub-second electrochemical measurements with two- photon microscopy in adult zebrafish with dopamine neurons that are genetically encoded with labels that either continually fluoresce (green fluorescent protein; GFP) or transiently fluoresce upon activation (GCaMP5). The long-term goal is to understand how changes in neuronal communication impact cognitive and motor function in neurological disease. The overall goal of this particular application is to develop and employ optical and neuronal stimulation techniques that enhance the utility of FSCV in understanding neuronal function in adult zebrafish. The model pathway, chosen because it is analogous to the mammalian mesolimbic pathway, originates in the posterior tubercle (PT) and extends to the dorsal nucleus of the ventral telencephalon (Vd). Aim 1 couples FSCV with two-photon imaging in GFP fish to test the hypothesis that electrically evoked dopamine release and uptake from individual neurons is heterogeneous. Aim 2 employs in vivo FSCV with optical measurement of neuronal firing in GCaMP5 fish to test the hypothesis that in vivo dopamine release events (`transients') in adult zebrafish differ in amplitude and frequency between neurons. The methods that will be developed and refined in this work will be the first to measure remotely evoked neurotransmitter release in the intact brain of adult zebrafish, ex vivo or in vivo. Once developed, this approach will allow the detailed characterization of numerous neurotransmitter release pathways in the zebrafish brain at the level of individual neuronal cell bodies within their complex, native environment. As a result, this work will facilitate studies in a wide range of neurological conditions, including Parkinson's disease, Alzheimer's disease, and Huntington's disease. Therefore, this proposed research is highly responsive to PA-18-358 (NINDS Exploratory Neuroscience Research Grant) as well as the NIH core mission of easing the burden of human disease.